Biological artificial nerve guide and method of making

ABSTRACT

A biological nerve guide for implantation into a human body is made by providing a natural animal tissue membrane, crosslinking and fixing the membrane, minimizing the antigens from the membrane, tanning the membrane, coupling an active layer to an inner surface of the membrane, cutting the membrane into a desired shape and size, positioning the cut membrane onto a rod-shaped mold so that the cut membrane assumes a cylindrical configuration, and attaching a spiral support to the outer surface of the cut membrane.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a medical prosthesis for humanimplantation, and in particular, to an artificial device for repairingneurons, such as a biological nerve guide.

2. Description of the Prior Art

Nerve tissues have regenerating power, and even the central nervoussystem has been discovered in recent years to possess regeneratingpower. However, nerve tissues are fragile and the regeneration speed isslow so that when neurons are damaged, natural regeneration and repairoften are unable to reconnect the nerve because of the slow growth rate.Also, the repair path is often blocked by the faster growing surroundingregenerated tissues or scar tissue.

To address these problems, some scientists have tried to utilize a guideto connect the two ends of a defective nerve to prevent the path frombeing blocked, and this guide is called a nerve guide. Some conventionalnerve guides are prepared from non-degradable materials so thatirritation from foreign matter was always present while regeneration ofnerve tissues was also adversely affected. Some of these conventionalnerve guides are prepared from degradable materials such as polylacticacid or polyglycolic acid, but their degraded products exhibit localizedacidity, adversely affecting the growth, the proliferation and themigration of nerve cells.

Other conventional nerve guides are produced from natural materials suchas animal blood vessels, but conventional glutaraldehyde is utilized inthe treatment process, resulting long-term residual toxicity and ratherpotent cellular toxicity while also adversely affecting the growth andproliferation of nerve cells. One of the serious drawbacks of thecurrent nerve guides is the thick guide wall which does not allow thepenetration of nutrients and the passage of blood supply, and the nervecells inside the guide cannot obtain enough nutrients for desirabledifferentiation and migration to repair damaged tissue.

Another conventional nerve guide is produced from degradable naturalmaterials such as animal collagen, but the mechanical properties such asflexibility, toughness, and kink resistance are not desirable. Anoticeable drawback is that the degradation speed is difficult to matchwith the speed of nerve tissue regeneration, so that the treatmentresult is often uncertain.

SUMMARY OF THE DISCLOSURE

It is an object of the present invention to provide a biological nerveguide having good biocompatibility.

It is another object of the present invention to provide a biologicalnerve guide that can be penetrated by nutrients and allows for effectiveflow of blood supply while capable of being absorbed.

It is another object of the present invention to provide a method ofpreparing a biological nerve guide that meets the objects set forthabove while overcoming the disadvantages described above.

In order to accomplish the objects of the present invention, the presentinvention provides a biological nerve guide-for implantation into ahuman body, the nerve guide made by the following method:

providing a natural animal tissue membrane;

crosslinking and fixing the membrane;

minimizing the antigens from the membrane;

tanning the membrane;

coupling an active layer to an inner surface of the membrane;

cutting the membrane into a desired shape and size;

positioning the cut membrane onto a rod-shaped mold so that the cutmembrane assumes a cylindrical configuration having an outer surface;and

attaching a spiral support to the outer surface of the cut membrane.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of an artificial biological nerve guideaccording to one embodiment of the present invention.

FIG. 2 is a cross-sectional view of the artificial biological nerveguide of FIG. 1.

FIGS. 3A-3C illustrate the surgical repair of a damaged nerve using thenerve guide of FIG. 1.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following detailed description is of the best presently contemplatedmodes of carrying out the invention. This description is not to be takenin a limiting sense, but is made merely for the purpose of illustratinggeneral principles of embodiments of the invention. The scope of theinvention is best defined by the appended claims.

The present invention provides a biological nerve guide having a thinguide body prepared from animal membrane materials treated bycrosslinked fixation with a non-aldehyde fixative, and having itsantigens minimized with reagents having strong hydrogen bonding. Aspiral support is formed by winding and immobilizing a long strip of theaforementioned membrane material around the guide wall.

Animal tissues are easily degraded or decomposed by microorganisms, sothat crosslinking and fixation with a fixative is required.Conventionally, glutaraldehyde is utilized as a fixative, butglutaraldehyde produces toxic radicals. Aldehydes undergo crosslinkingwith proteins through the acetal reaction and toxic aldehydes arereleased when the crosslinked products are degraded, so that productsfixed with an aldehyde have long-term residual toxicity. Whennon-aldehyde fixatives such as epoxides, diacyl diamides, diisocyanates,polyethylene glycol or carbodiimides are utilized as fixatives in placeof aldehydes, this toxicity problem can be minimized or even eliminated.For example, when an epoxide is utilized to replace aldehyde-typefixatives, a ring-opening/crosslinking reaction occurs readily becauseepoxides are unstable, but the crosslinking product can be made verystable and not easily degraded by controlling the reaction condition. Itis slowly degraded into polypeptides and amino acids and absorbed onlywhen tissue growth and regeneration begin to devour it by secretingkallikrein, fibrinolysin and glucocorticoid hormone to help collagenasein the degradation. Such kind of passive degradation and tissueregeneration are occurring synchronously which is beneficial to tissueregenerative repair while having no residual toxicity of aldehydes.According to modern immunological theory, the antigenicity of animaltissues stems mainly from active groups located at specific sites and inspecific conformations, and these active groups include —OH, —NH2, —SH,etc. The specific conformations result mainly from some specifichydrogen bonding formed by spiral protein chains. The specific sites andconformations are called antigen determinants. One or more activereagents (e.g., acid anhydrides, acyl chlorides, amides, epoxides, etc.)that react readily with these groups are utilized to bond with and blockthese groups when treating animal tissues so that the antigens can beeffectively minimized or eliminated. Simultaneously, reagents withstrong hydrogen bonding (e.g., guanidine compounds) are utilized toreplace the hydrogen bonding that gives the specific configurations sothat the configurations are altered and the antigenicity is effectivelyeliminated.

The wall of the nerve guide of the present invention is a thinpermeable, semi-transparent membrane for insuring easy penetration ofnutrients and microveins so that the need for regeneration of the nervetissues is provided. A spiral support is provided on the guide wall toprovide sufficient supporting power for the body of the guide, and tomaintain a space for the path required for regeneration of nervetissues. In addition, the winding, stretching and mechanicalcompatibility of the spiral support facilitate nerve repair at motorparts. Both the guide body and the spiral support are prepared usinganimal tissues as the starting materials, and the main component iscollagen with a small quantity of glycoproteins, and they can bedegraded to amino acids and polypeptides which can be absorbed by humanbodies.

Tanning

The present invention uses an additional cross-linking method and aprotein grafting method as a tanning process to improve the mechanicalstrength and toughness of the tissue. In this regard, a piece of animalmembrane tissue usually provides poor mechanical properties (afterharvesting). As used herein, “mechanical properties” means strength,toughness, rigidity and modulus. Both cross-linking and protein graftingcan alter the mechanical properties of the tissue collagen (protein)matrix. Although cross-linking and protein grafting are common methodsthat are used to improve the mechanical properties of high polymers, itis still important to carefully determine the selection of reagents aswell as the reaction conditions because protein can often be denatured.The length, density and distribution of cross-linkage are properlydesigned to ensure the stability of the tissue material and mechanicalproperty.

For example, the molecular chain length of the crosslinking agentdetermines the cross-linking length. A longer chain results in bettermaterial flexibility. However, larger molecular chains are moredifficult to penetrate into the collagen matrix. For example, whenselecting an epoxy compound as the cross-linking agent, the molecularchain is preferably 4-8 hydrocarbons. The cross-linking densitydetermines the cross-linking degree. Denser cross-linking results inbetter material stability, but denser cross-linking (especially whencombined with a shorter molecular chain) can introduce a higher localstress in the material. A relatively uniform distribution of thecross-linking is ideal, but is usually difficult to obtain. Utilizing alower concentration of the cross-linking solution, under a lowertemperature, longer reaction duration, and repeating a few more timeswith the same reaction can often yield better results. As an example,when using an epoxy compound as the cross-linking agent as described inU.S. Pat. No. 6,106,555, good material stability, good flexibility,toughness and strength can be obtained by picking 4-8 hydrocarbon atomchain, with a concentration of 0.1 to 2%, under 4 to 24 degrees Celcius,reaction for 3-10 days, and repeating 2 to 5 times.

The chemical reagents can be the same as those described herein for usewith tissue fixation. The protein grafting process can further improvethe tissue's mechanical strength, toughness, rigidity and modulus.Protein grafting requires a large amount of polymer chains so that thenature of the protein structure can be changed substantially. Some highpolymers can be grafted into collagen molecules by means ofpolycondensative primers. In order to avoid introducing hazardoussubject matter into the human body, it is preferable to usebiodegradable high polymers as the grafting agents, such as polyglycolicacid (PGA), polylactic acid (PLA) and others. These biodegradablepolymers can be metabolized in the host environment through atracarboxylic acid cycle just like for carbohydrates or fat metabolism.After such an extensive protein modification, up to 25 kGy gamma raysterilization can be applied without adversely affecting the mechanicalproperty of the tissue material. The total amount of protein graftingcan be controlled optimally.

Active Layer

The surface of the nerve guide can also include an active layer. Thisactive layer can contain a polypeptide or glycosaminoglycan. One exampleof the polypeptides is the polypeptide obtained from the condensation of16 lysines (K16), glycine (G), arginine (R), asparagic acid (D), serine(S), proline (P) and cysteine (C), and said glycosaminoglycan ishyaluronic acid, chondroitin sulfate, dermatan sulfate, heparin,acetylheparin sulfate or keratan sulfate. These polypeptides orglycosaminoglycans exhibit a broad-spectrum adherence and enrichingeffect for growth factors or activate undifferentiated cells to performoriented differentiation so that they are capable of exercising thefunction of inducing regenerative repair of organic tissues.

Materials

The body of the nerve guide, and the spiral support, can be made usinganimal intestinal membrane, pericardium, pleura or omentum.

Method

A method of preparing the biological nerve guide according to thepresent invention comprises the following steps:

1. Selection and cleaning of materials: Fresh animal membrane tissuesare collected and sterilized with benzalkonium chloride orchlorhexidine, and trimmed to remove excessive impurities and irregularparts. The required membrane materials are obtained by taking andcleaning the neat and tough membrane materials.

2. Defatting: Fats and fat-soluble impurities in the membrane areextracted with an organic solvent.

3. Crosslinking fixation: The collagen molecules in the membrane arecrosslinked and fixed with a non-aldehyde fixative.

4. Minimize antigens: The specific active group, namely —OH or —NH₂ or—SH, in the proteins of the membrane is blocked with an active reagentand the specific hydrogen bonding in the spiral chains of the proteinsin the membrane is replaced by using a reagent having strong hydrogenbonding.

5. Tanning process: First, the preformed polymers are produced frommonomers by synthesis. Second, the membrane is dehydrated with alcohol.Third, the preformed polymers are then grafted into collagen moleculesby means of polycondensative primers. When using PGA as the graftingreagent, a small amount of glycolide may be used as the polycondensativeprimer. When using PLA as the grafting reagent, a small amount oflactide may be used as the polycondensative primer.

For example, using PLA as the protein grafting agent, the process couldtake 30-50 mg of lactide and dissolve it in 1000 ml of chloroform. 2-3grams of triisobutyl aluminum can be added as the composite catalyst,and this solution can be stir-mixed for one to two hours under atemperature of 40-60 degrees Celcius. 100 ml of a 0.1N NaOH solution isthen added and stir-mixed for 30-60 minutes to destroy the catalyst.Then take away the separated water layer (with catalyst) and have thepreformed polymers ready. Immerse the dehydrated membrane into thepreformed polymer solution. Add 0.1 to 2 g of lactide and 0.5 to 5 g ofproprionic anhydride as an initiation catalyst and then stir-mix for 2-4hours under a temperature of 34 to 40 degrees Celcius. Take out themembrane and put it into chloroform to clean away the residual preformedpolymers. After rinsing with saline, the membrane is then immersed intosaline for 12 to 24 hours to recover the water content. The membrane isnow ready for the next processing step.

6. Coupling of active layer: An active surface layer is coupled to thesurface of the guide body using a coupling agent. The active surfacelayer has an active component such as a polypeptide orglycosaminoglycan. Specifically, the surface of the membrane material iscoupled with a polypeptide or glycosaminoglycan capable of adhering togrowth factors to form an active surface layer.

7. Preparation of the nerve guide: The membrane material is glued on arod-shaped mold with medical gel to form a guide body. Separately, thesame (or different) membrane material is cut to a specific width andthen glued on the surface of the guide body by winding spirally at agiven distance in multiple layers to form a spiral support having aparticular supporting power. Next, the mold is removed to yield thefinal product.

Fixative

The fixative applied in step 3 of the above method can be a reagent thatcrosslinks easily with protein molecules and is one or two reagentsselected from epoxides, diacyl diamides, diisocyanates, polyethyleneglycol or carbodiimides. This fixative may be an epoxy compound that hasa hydrocarbon backbone, that is water-soluble, and which does notcontain an ether or ester linkage in its backbone. This fixative isdescribed in U.S. Pat. No. 6,106,555, whose entire disclosure isincorporated by this reference as though set forth fully herein.Examples include an epoxide, a diamide, a diisocyanate, a polyethyleneglycol, or a carbodiimide, in that the epoxide may be a monocyclicepoxide, or a bicyclic epoxide, or it may be a low poly(epoxide) (suchas low poly(ethylene oxide), poly(propylene oxide) or a glycidyl ether).The epoxide may be a monocyclic epoxide

or a dicyclic epoxide

where R═H, C_(n)H_(2n+1)-, n=0-10, and may also be a lower polyepoxidesuch as polypropylene oxide.

Active Reagents

The active reagents in step 4 of the above method may be low molecularweight organic acid anhydrides, acyl chlorides, acyl amides, monocyclicoxides or epoxide, and the reagents having strong hydrogen bonding powerare guanidine compounds.

Coupling Agent for Active Layer

The coupling agent utilized for coupling the polypeptide in step 6 ofthe above method may be a diacyl diamide, diacid anhydride, diepoxide orother bifunctional reagents capable of carrying out condensation with—NH₂, —OH and —COOH.

The present invention provides the following advantages. The finalproduct is prepared by using natural biological materials such as animaltissues as the starting materials so that there is no immunogenicity,and minimal rejective reaction, while having excellent tissuecompatibility and being capable of inducing division, proliferation andmigration of nerve cells and promoting regeneration of nerve tissues.Pathway space required for the growth of nerve tissues is provided sothat nutritional need for the growth of nerve tissues is suppliedthrough penetration of nutrients and in-growth of blood vessels, therebycreating an excellent microenvironment for regenerative repair of thenerve tissues. After repair of the nerve tissues is completed, thebiological nerve guide can be degraded and absorbed such that it is notpresent as a foreign matter.

EXAMPLE 1

Referring to FIGS. 1 and 2, fresh porcine membrane materials such aspericardium, omentum, pleura, diaphragm or small intestine membrane areexcised, and the fatty materials and loose fibrous tissues are carefullyremoved, to trim the tough membrane to be as thin as possible. Then, themembrane is washed, cleaned and rinsed with water, and then the fats andfat-soluble impurities in the membrane materials are extracted using anorganic solvent. The membrane will be used for the guide body 1 and thespiral support 2 shown in FIGS. 1-2.

Next, the solvent is removed and crosslinking fixation is conductedusing a carbocyclic oxide.

After washing and freeze-drying, reaction with acetic anhydride orbutyric anhydride is conducted to block the antigen groups, and themembrane is treated with Tris buffer solution of guanidine hydrochlorideto alter the specific conformations of the antigens.

Polyglycolic acid prepolymer is then grafted on the collagen moleculesto strengthen the durability using an acid anhydride as a condensationagent.

A diacid intramolecular anhydride is then utilized as a bifunctionalcoupling agent to couple the polypeptide obtained by condensing 16lysines (K16), glycine (G), arginine (R), asparagic acid (D), serine(S), proline (P) and cysteine (C) or a glycosaminoglycan on the surfaceof the membrane material to form the active surface layer 3 on whatwould be the inner surface of the cylindrical guide body 1.

At this point, the membrane material is cut according to the desiredspecifications, and then glued on a rod-shaped mold to form the guidebody 1 using medical gel. Separately the membrane material is cut into along strip (e.g., about 0.5-2.0 mm wide) and rolled around and glued onthe outer surface of the guide body 1 in a spiral manner to form thespiral support 2. Multiple layers of the spiral support 2 can beprovided (by gluing) to increase the diameter of the nerve guide or toadd another piece of spiral component until the required supportingpower is attained. The nerve guide is then removed from the mold,washed, sterilized and then packaged by sealing with physiologicalsaline solution being used as a preservative solution.

FIGS. 3A-3C illustrate how the nerve guide of FIGS. 1-2 can be used inthe surgical repair of a damaged nerve N. FIG. 3A shows a nerve N thathas been damaged (e.g., severed). As shown in FIG. 3B, the ends of thedamaged nerve N can be inserted into the cylindrical bore of the nerveguide of FIG. 1, with the nerve guide serving as a connector. Sutures 4can be applied to suture or attach the ends of the nerve guide to thedamaged nerve.

While the description above refers to particular embodiments of thepresent invention, it will be understood that many modifications may bemade without departing from the spirit thereof. The accompanying claimsare intended to cover such modifications as would fall within the truescope and spirit of the present invention.

1-20. (canceled)
 21. A biological nerve guide for implantation into ahuman body, comprising: a cylindrical body isolated from its host thathas a substrate that has: (i) been fixed with crosslinking reagents,(ii) residual specific active groups in protein molecules of thesubstrate that have been blocked by at least one active reagent afterfixation by the crosslinking reagents, (iii) specific conformation ofprotein molecules of the substrate altered by a reagent with stronghydrogen bonding power, and (iv) an active layer coupled thereto, theactive layer including either a polypeptide or a glucosaminoglycan thathas the ability to adhere growth factors after implantation; and aspiral support that is attached to the outer surface of the cylindricalbody.
 22. The guide of claim 21, wherein the substrate is fixed by anepoxy compound, a diamide, a diisocyanate, or a carbodiimide.
 23. Theguide of claim 21, wherein the at least one active reagent to blockspecific active groups in the protein molecules of the substrate can beacid anhydrides, acid chlorides, or acylamides.
 24. The guide of claim21, wherein the reagent with strong hydrogen bonding power is aguanidine compound.
 25. The guide of claim 22, wherein the epoxycompound has a hydrocarbon backbone, that is water-soluble and whichdoes not contain an ether or ester linkage in its backbone.
 26. Theguide of claim 21, wherein the spiral support is cut from the samematerial as the substrate.
 27. The guide of claim 21, wherein themembrane has been tanned.